In mammalian cells, the structural integrity of the nucleus is conferred by A- and B-type lamins, a meshwork of proteins that lines the nucleoplasmic face of the nuclear envelope and forms the nuclear lamina. Mutations scattered along LMNA, which encodes A-type lamins, as well as mutations within other nuclear envelope proteins are associated with a broad range of human diseases, collectively called laminopathies. The molecular etiology of these diseases remains unknown. The recent characterization of the LINC complex, an evolutionary-conserved protein complex that bridges the inner and outer membranes of the nuclear envelope and physically connects the nuclear lamina to the cytoskeleton of mammalian cells, suggests that the nucleus is directly tethered to the cytoskeleton. This structural linkage is crucial for at least two reasons: (1) it is important to maintain nuclear morphology per se and (2) it allows mechanical force to be exerted upon the nucleus to move it within the cell, a process vitally important in muscle and nerve cell development, for example. At the center of the LINC complex is the interaction between SUN domain containing proteins of the inner nuclear membrane and KASH-peptide-containing proteins of the outer nuclear membrane. SUN and KASH interact directly and specifically in the perinuclear space. In many of the laminopathies, including the severe muscle disease, Emery-Dreifuss muscular dystrophy, neurological disorders such as cerebellar ataxia, and progeria the SUN-KASH interaction within the LINC complex is lost, resulting in severe consequences for nuclear morphology and positioning. In humans, at least five LINC complexes exist, each containing a different pair of SUN- and KASH-containing proteins. Signaling between these components is poorly understood. At the center of this proposal is the elucidation of the structure and function of the SUN-KASH interaction using biochemical and X-ray crystallographic tools. The atomic structure of the SUN-KASH complex should provide valuable insight into the regulation of this interaction. Despite the importance of the SUN-KASH complex for human physiology and pathology, its structure is still unknown. This can largely be attributed to the problems with obtaining sufficient quantities of the proteins for structural studies. We have developed a method to overcome these obstacles and are now able to pursue quantitative structure-function analysis. We anticipate that this project will shed light on the biophysical principles behind the nucleo-cytoskeletal connection established through LINC complexes. We will identify by X-ray crystallography the specific molecular details that regulate this conserved interconnection. In parallel, we will perform detailed biochemical and mutational analysis of the SUN-KASH complex. This study has the potential to enable the structural and molecular characterization of the entire LINC complex, including all other components. In consequence, this project should establish new drug targets for the cure of a broad array of laminopathies. PUBLIC HEALTH RELEVANCE: Muscular dystrophies collectively have a high impact on health, affecting tens of thousands of people in the United States alone. Emery-Dreifuss muscular is characterized by wasting of certain muscles, joint deformities and life-threatening heart problems that can result in premature and sudden death. There is currently no definitive therapy for Emery-Dreifuss muscular dystrophy or related diseases; therefore, the work in this project is designed to identify molecular targets that can lead to treatments for patients.